The five muscarinic acetylcholine receptors (m1-m5) are members of a superfamily of plasma membrane receptors which regulate cellular activity via coupling to heterotrimeric G proteins. We have used the muscarinic receptors as model systems to study how G progein-coupled receptors function at a molecular level. The molecular mechanisms involved in receptor assembly, ligand binding, and G protein coupling were studied by using a combined pharmacologic/molecular genetic approach. Functional analysis of a series of hybrid m2/m5 muscarinic receptors showed that the three dimensional structure of muscarinic receptors (and, most likely, other G protein-coupled receptors) is similar to that of bacteriorhodopsin and that direct molecular interactions between TM I and TM VII are required for proper receptor folding. Mutational analysis of the m3 muscarinic receptor demonstrated that a TM VI Asn residue (Asn507) which is characteristic for the muscarinic receptor family plays a specific role in the binding of certain subclasses of muscarinic antagonists including atropine-like agents and pirenzepine. Replacement of Asn507 by Ala, Ser, or Asp led to mutant m3 receptors with drastically reduced binding affinities (up to 28,000-fold) for these ligands. Previous studies have shown that the N-germinal segment of the third intracellular loop (i3) is critically involved in determining the G protein coupling properties of the individual muscarinic receptor subtypes. Using a novel insertion mutagenesis approach, we could demonstrate that this region forms an amphophilic alpha-helical extension of TM V and that one side of this helix (most likely the hydrophobic one) represents a major G progein recognition surface. Systematic mutational modification of the N-terminal segment of the i3 loop of the rat m3 muscarinic receptor showed that a tyrosine residue (Tyr254) located at the beginning of this domain is essential for efficient activation of Gq (mediated by the m1, m3, and m5 muscarinic receptors). These studies should eventually lead to a detailed structural model of the ligand-receptor-G protein complex which should provide a rational basis for the development of novel muscarinic drug.